Abstract Zika virus (ZIKV), a flavivirus, is the cause of the recent pandemic of infections across both North and South America often associated with serious neurological damage, including congenital microcephaly in newborns and Guillain-Barr syndrome in adults. According to the CDC, almost 5% of women who were infected with ZIKV during pregnancy give birth to babies with severe birth defects, including microcephaly and brain abnormalities. Long-term damage caused by ZIKV infection to newborns of women who were infected with ZIKV during pregnancy is still unclear. Despite the significant medical need for prophylactic or therapeutic treatments, no vaccines or drugs have been approved for ZIKV infections. The overall goal of this project is to address this significant unmet medical need by developing novel scaffolds of small molecule inhibitors of ZIKV for prophylactic and therapeutic use. The strategy of this proposal is to optimize the potency, selectivity and drug-like properties of a novel phenylcarbamoyl (PCA) series of compounds that we previously demonstrated to inhibit ZIKV infections in culture and to develop resulting prioritized analogs into anti-ZIKV drugs. PCA analogs exhibit IC50 values ?0.5 M vs ZIKV in culture, are tolerated at doses up to a solubility-limited dose of 30 mg/kg in mice via IP administration, and exhibit murine plasma availability >10-fold above the IC50 for up to 6 hrs. The chemical optimization of this proposal is designed to maximize the potency, selectivity, and dug-like properties to generate in vivo-validated lead compounds with efficacy against Asian, African, and South American strains of ZIKV. The approach is to leverage our experience in chemical synthesis and in cell-based infection, viral entry and viral replication assays to optimize this PCA scaffold. In Phase I of this project, in Aim 1, SAR-based chemical optimization will be applied to maximize the anti-ZIKV potency and selectivity of the PCA inhibitor series. In Aim 2, the activity of analogs will be evaluated versus infectious ZIKV strains isolated from Uganda, South Asia, and South America, and prioritized based on their activity, selectivity, and solubility. The mechanism of action of resulting prioritized compounds will be investigated in a variety of cellular assays for ZIKV and other RNA viruses to determine the antiviral spectrum and verify selective antiviral activity. In Aim 3, analogs will be prioritized by their in vitro ADME properties, formulations for IP administration will be developed, and in vivo tolerability and PK profiles of PCA analogs with high potency and selectivity will be determined. In Aim 4, prioritized PCA analogs with favorable in vitro ADME properties and in vivo PK profiles will be evaluated for efficacy against ZIKV infection in a mouse model to define in vivo-validated leads. In Phase II, lead compounds will be optimized further to improve pharmacokinetic properties as well as efficacy and selectivity to generate a preclinical candidate and a backup compound.